The dynamics of secretory vesicles in living hyphae of the pathogen Ustilago maydis.

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Filamentous fungi are an evolutionarily successful group of organisms of enormous ecological importance as symbionts in mycorrhizal interactions with plants and decomposer of plant debris. They serve in industrial production of proteins and as pathogens pose a threat to public health and agriculture. The basic unit of a filamentous fungus is the hypha; this usually consists of a chain of elongated cells that grow by expansion at the tip, a process called tip growth. This mode of growth allows the invasion of tissue and substrate. Tip growth requires a continuous supply of newly synthesised membranes, proteins and cell wall precursors to the hyphal tip in a process named secretion. The carriers for these supplies are secretory transport vesicles that are taken to the tip where they fuse with the plasma membrane. It is important in the context of this grant application to note that our current view of secretion is that post-Golgi vesicles travel uni-directionally to the hyphal tip where they fuse with the plasma membrane, thereby delivering membranes and proteins. The mechanism by which secretory vesicles are delivered to the hyphal tip is not clear. In analogy to other polarised cells, such as animal neurons, it is assumed that microtubules and kinesin motors mediate long-distance transport of secretory vesicles, whereas short range motility at the plasma membrane is mediated by another system, consisting of myosins and filamentous actin (F-actin). Indeed, recent studies on numerous filamentous fungi, including the plant pathogen Ustilago maydis, demonstrated that microtubules, kinesins, F-actin and myosins are essential for hyphal growth. However, most conclusions from these data are speculative. This due to the fact that motility of secretory cargo was never visualised and no systematic studies on the whole repertoire of motors in a cell have been undertaken. In this project we will address this challenge. We have developed a microscopic setup that allows us to visualise the motility of individual chitin synthase-containing vesicles. We will label numerous other secretory proteins, including secreted enzymes, and will monitor their delivery to the growing hyphal tip. In co-localisation studies using red and green fluorescent proteins we will determine whether cargo travels in the same or different transport vesicles, thereby elucidating the pathways of secretion. We will determine the cytoskeletal elements that underlie vesicle motility and address the role of all kinesin and myosin motors in secretion by making use of existing mutant protein constructs. Finally, we will further investigate the reason for the bi-directional motility of chitosomes. This behaviour is unexpected and we will investigate whether it is a general feature of secretory vesicles. Subsequently, we will use photoactivatable fluorescent proteins to characterise this motility in order to get an insight into the reason for this phenomenon. In summary, we will combine molecular genetics and life cell imaging to: (1) determine the pathways of secretion in the hyphal cell, (2) address the role of 10 kinesins and 4 myosins in motility of secretory vesicles, (3) characterise the bi-directional motility of secretory vesicles in order to get to an understanding the logic behind this behaviour. The expected outcome of this project will be novel insights into the secretory pathway in filamentous fungi. We will provide a comprehensive understanding of the pathways by which cargo reaches the hyphal tip and of the role of kinesins and myosins in delivery of secretory vesicles. If it is discovered that bi-directional motility of secretory vesicles is a general feature, the current paradigm for secretion will have to be modified. This project will therefore be of fundamental interest to all aspects of fungal research, but it will be of particular importance in understanding fungal pathogenicity and industrial production of recombinant proteins.